Citation: Chunchun Meng, Yunxiu Huang, Zaib Ur Rehman, Wen Hu, Chuanfeng Li, Ruiying Liang, Zongyan Chen, Kaijie Song, Tianchao Wei, Guangqing Liu. Development of an MCA-Based Real Time RT-qPCR Assay for the Simultaneous Detection and Differentiation of Duck Hepatitis A Virus Types 1 and 3 .VIROLOGICA SINICA, 2020, 35(5) : 666-669.  http://dx.doi.org/10.1007/s12250-020-00211-8

Development of an MCA-Based Real Time RT-qPCR Assay for the Simultaneous Detection and Differentiation of Duck Hepatitis A Virus Types 1 and 3

  • Corresponding author: Guangqing Liu, liugq@shvri.ac.cn
  • Received Date: 16 October 2019
    Accepted Date: 03 March 2020
    Published Date: 08 April 2020
    Available online: 01 October 2020

  • 加载中
    1. Cha SY, Roh JH, Kang M, Kim B, Jang HK (2013) Isolation and characterization of a low pathogenic duck hepatitis A virus 3 from South Korea. Vet Microbiol 162:254-258
        doi: 10.1016/j.vetmic.2012.11.023

    2. Chen LL, Xu Q, Zhang RH, Yang L, Li JX, Xie ZJ, Zhu YL, Jiang SJ, Si XK (2013) Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duck hepatitis A virus type 1 and type 3 in ducklings. J Virol Methods 192:12-17
        doi: 10.1016/j.jviromet.2013.04.012

    3. Chen X, Chen Y, Liu C, Li X, Liu H, Yin X, Bai X, Ge M, Chen H, Liu M, Du Y, Fan G, Zhang Y (2019) Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3. PLoS ONE 14:e0219750
        doi: 10.1371/journal.pone.0219750

    4. Ding C, Zhang D (2007) Molecular analysis of duck hepatitis virus type 1. Virology 361:9-17
        doi: 10.1016/j.virol.2007.01.007

    5. Doan HT, Le XT, Do RT, Hoang CT, Nguyen KT, Le TH (2016) Molecular genotyping of duck hepatitis A viruses (DHAV) in Vietnam. J Infect Dev Ctries 10:988-995
        doi: 10.3855/jidc.7239

    6. Gan Y, Huang C, Wang X, Chen S, Li J (2014) Epidemiological investigation of duck hepatitis A virus (DHAV) isolated from Sichuan Basin by RT-PCR disclose the existence of mixed infection and the feasibility of DHAV-A evolved from C80 strain. Pak Vet J 34:356-360

    7. Hu Q, Zhu D, Ma G, Cheng A, Wang M, Chen S, Jia R, Liu M, Sun K, Yang Q (2016) A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3. J Virol Methods 236:207-214
        doi: 10.1016/j.jviromet.2016.07.011

    8. Huang Q, Yue H, Zhang B, Nie P, Tang C (2012) Development of a real-time quantitative PCR for detecting duck hepatitis a virus genotype C. J Clin Microbiol 50:3318-3323
        doi: 10.1128/JCM.01080-12

    9. Kim MC, Kwon YK, Joh SJ, Lindberg AM, Kwon JH, Kim JH, Kim SJ (2006) Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae. J Gen Virol 87:3307-3316
        doi: 10.1099/vir.0.81804-0

    10. Kim MC, Kwon YK, Joh SJ, Kim SJ, Tolf C, Kim JH, Sung HW, Lindberg AM, Kwon JH (2007) Recent Korean isolates of duck hepatitis virus reveal the presence of a new geno- and serotype when compared to duck hepatitis virus type 1 type strains. Arch Virol 152:2059-2072
        doi: 10.1007/s00705-007-1023-0

    11. Lin SL, Cong RC, Zhang RH, Chen JH, Xia LL, Xie ZJ, Wang Y, Zhu YL, Jiang SJ (2016) Circulation and in vivo distribution of duck hepatitis A virus types 1 and 3 in infected ducklings. Arch Virol 161:405-416
        doi: 10.1007/s00705-015-2648-z

    12. Liu M, Fanyi M, Li XJ, Zhang Z, Liu S, Zhang Y (2011) Goose haemorrhagic hepatitis caused by a new subtype duck hepatitis type 1 virus. Vet Microbiol 152:280-283
        doi: 10.1016/j.vetmic.2011.05.015

    13. Soliman M, Alfajaro MM, Lee MH, Jeong YJ, Kim DS, Son KY, Kwon J, Choi JS, Lim JS, Choi JS, Lee TU, Cho KO, Kang MI (2015) The prevalence of duck hepatitis A virus types 1 and 3 on Korean duck farms. Arch Virol 160:493-498
        doi: 10.1007/s00705-014-2264-3

    14. Toth TE (1969) Studies of an agent causing mortality among ducklings immune to duck virus hepatitis. Avian Dis 13:834-846
        doi: 10.2307/1588590

    15. Tseng CH, Knowles NJ, Tsai HJ (2007) Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus. Virus Res 123:190-203
        doi: 10.1016/j.virusres.2006.09.007

    16. Tseng CH, Tsai HJ (2007) Molecular characterization of a new serotype of duck hepatitis virus. Virus Res 126:19-31
        doi: 10.1016/j.virusres.2007.01.012

    17. Wen X, Zhu D, Cheng A, Wang M, Chen S, Jia R, Liu M, Sun K, Zhao X, Yang Q, Wu Y, Chen X (2018) Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015. Transbound Emerg Dis 65:10-15
        doi: 10.1111/tbed.12741

    18. Yang M, Cheng A, Wang M, Xing H (2008) Development and application of a one-step real-time Taqman RT-PCR assay for detection of Duck hepatitis virus type1. J Virol Methods 153:55-60
        doi: 10.1016/j.jviromet.2008.06.012

    19. Yugo DM, Hauck R, Shivaprasad HL, Meng XJ (2016) Hepatitis virus infections in poultry. Avian Dis 60:576-588
        doi: 10.1637/11229-070515-Review.1

    20. Zhang R, Xia L, Chen J, Gong Y, Zhang L, Li P, Liu H, Xie Z, Jiang S (2017) Molecular epidemiology and genetic diversity of duck hepatitis A virus type 3 in Shandong province of China, 2012-2014. Acta Virol 61:463-472
        doi: 10.4149/av_2017_409

  • 加载中

Figures(1) / Tables(1)

Article Metrics

Article views(3969) PDF downloads(29) Cited by()

Related
Proportional views

    Development of an MCA-Based Real Time RT-qPCR Assay for the Simultaneous Detection and Differentiation of Duck Hepatitis A Virus Types 1 and 3

      Corresponding author: Guangqing Liu, liugq@shvri.ac.cn
    • 1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China
    • 2. College of Animal Science and Technology, Guangxi University, Nanning 530005, China

    Abstract: 

    • Dear Editor,

      Duck virus hepatitis (DVH) is a significant concern in the duck industry as the disease causes a highly contagious infection in young ducklings that is often associated with liver necrosis, hemorrhage, and high mortality (Yugo et al. 2016). Duck hepatitis virus (DHV) was first described in 1949 on Long Island in the United States. Subsequent, outbreaks have been reported in England, Canada, Germany, Japan and elsewhere (Toth 1969). DHV is associated with at least two RNA viruses, duck hepatitis A virus (DHAV) and duck astrovirus (DAstV); however, no antigenic relationships have been identified between these two viruses (Yugo et al. 2016). DHAV is the primary causative agent of DVH. As the only member of the genus Avihep-atovirus, in the Picornaviridae family, DHAV has a linear, single-stranded positive-sense RNA genome. The genomic organization of DHAV is analogous to that of other picornaviruses with one large open reading frame (ORF) that encodes a polyprotein precursor, that is preceded by a 5'-untranslated-terminal-region (UTR) and followed by 3'- UTR (Tseng et al. 2007). Based on systematic phylogenetic analyses and neutralization assays, DHAVs have been classified into three serotypes: the classical serotype 1 (DHAV-1) (Kim et al. 2006; Ding and Zhang 2007; Tseng et al. 2007), the second serotype that has only been reported in Taiwan Province of China (DHAV-2) (Tseng and Tsai 2007), and the third serotype that was first reported in South Korea (DHAV-3) (Kim et al. 2007). DHAV-3 also accounts for an increasing proportion of DHV pathogens in China (Liu et al. 2011; Zhang et al. 2017; Wen et al. 2018), South Korea (Cha et al. 2013; Soliman et al. 2015) and Vietnam (Doan et al. 2016).

      Previous studies have demonstrated that there is no cross-neutralization between DHAV-1 and DHAV-2 (Tseng and Tsai 2007) and that there is only limited crossneutralization between DHAV-1 and DHAV-3 (Kim et al. 2007). In many duck farms, DHAV-1 and DHAV-3 infections often occur at the same time, and their clinical symptoms and pathological developments are not easy to distinguish. Therefore, it is necessary to establish a rapid diagnostic technique for the simultaneous detection and differentiation of DHAV-1 and DHAV-3.

      In the present study, we developed a SYBR Green I realtime RT-qPCR assay for the simultaneous detection and differentiation of DHAV-1 and DHAV-3 using amplicon melting curve analysis (MCA) of only one primer pair. The parameters were optimized, and the sensitivity, specificity and repeatability were evaluated. The diagnostic application of the assay was assessed on clinical samples.

      At total of 77 published whole genome sequences (54 from DHAV-1 and 23 from DHAV-3) were retrieved from the GenBank database and aligned using the DNAStar (DNASTAR, Inc., United States) program. A highly conserved region located in the 5'-UTR was used to design primers for the detection and differentiation of DHAV-1 and DHAV-3. The optimized primers (5' UTR-F: 5' GTTGTGAAACGGATTACCGGTAGT 3'; 5' UTR-R: ACTCGACCAGCCGCGACCCTAT), yielded an expected PCR product of 202 base pairs that could bind all the DHAV-1 and DHAV-3 strains. In addition, a significant difference in the G+C content in the interior regions of the DHAV-1 and DHAV-3 sequences was used to facilitate an effective MCA. The SYBR Green-I RT-qPCR assay was conducted using the one step SYBR PrimeScript RT-PCR kit (Takara, China) following instructions in the kit manual.

      To test the assay's sensitivity, two standard curves were generated with tenfold serially diluted DHAV-1 and DHAV-3 standard plasmids with concentrations of 1.0 × 101-1.0 × 109 copies/iL respectively. All the samples were tested in duplicate with three independentruns. As standard curve analysis demonstrated that when the copy numbers were in the range of 102 to 109 copies/^L, the efficiency of the qPCR reaction for DHAV-1 and DHAV-3 was 100% and 98%, respectively. The correlation coefficient R2 for the linear regression equation of DHAV-1 and DHAV-3 was 0.999 and 0.997, respectively (Fig. 1A and 1B). Furthermore, the detection limits forboth DHAV-1 and DHAV-3 were 10 copies/μL. The melting curves both of DHAV-1 and DHAV-3 displayed single peaks and had consistent Tm values of approximately 86.15 ℃ and 83.86 ℃, respectively(Fig. 1C).

      Figure 1.  Simultaneous detection and differentiation of duck hepatitis A virus type 1 and 3 through melting curve analysis by one pair of primer. A, B Standard curves of real-time PCR for DHAV-1 and DHAV-3. Tenfold diluted standard plasmid of DHAV-1 and DHAV-3 tested with same pair of primer. Regression lines between the Ct values and the input concentrations of DHAV-1 (A) and DHAV-3 (B) plasmid DNA using SYBR Green I real-time PCR. C Melting curve analysis of DHAV-1 and DHAV-3. Amplification melting curve and melting temperature values (Tm) after SYBR Green I realtime PCR followed by melting curve analysis of DHAV-1 and DHAV-3. D Specificity of realtime PCR assay. The specific fluorescent signals were detected from cDNA of DHAV-1 and 3, and the dissociation curves showed that there were two specific product peaks for DHAV-1 and DHAV-3, respectively, but no specific amplification for negative control (DAstV, AIV, DRV, DEV and DPV).

      To test the assay's specificity, DAstV, avian influenza virus (AIV), duck reovirus (DRV), duck virus enteritis virus (DEV), and duck parvovirus (DPV) genomic cDNA or DNA are used as templates in the SYBR Green I real time RT-qPCR assay, using the aforementioned reaction conditions (Fig. 1D). No consistently positive signals were observed for any of these samples, suggesting that the realtime RT-qPCR assay was specific for DHAV.

      To assess the reproducibility of the RT-qPCR assay, the Ct values for two standard plasmids (DHAV-1 and DHAV-3, 103 to 107 plasmid copies/^L) were detected in inter and intra-assays that were performed in triplicate. As shown in Table 1, the coefficients of variation (CV) of the CT values were less than 5%. Specifically, the CV of the intra- and inter-assays ranged from 0.47% to 0.69% and from 0.51% to 1.5% for the DHAV-1 plasmids and, from 0.70% to 1.40% and from 0.34% to 0.87% for the DHAV-3 plasmids, respectively.

      No. of DNA copies Intra-assay Inter-assay
      Ct (mean±SD) CV (%) Ct (mean±SD) CV (%)
      DHAV-1
        1.0 × 107 18.43 ± 0.31 0.51 18.51 ± 0.087 0.47
        1.0 × 105 25.07 ± 0.20 0.79 25.19 ± 0.24 0.95
        1.0 × 103 28.40 ± 0.44 1.5 28.62 ± 0.20 0.69
      DHAV-3
        1.0 × 107 18.28 ± 0.16 0.87 18.21 ± 0.26 1.4
        1.0 × 105 25.29 ± 0.15 0.59 25.22 ± 0.12 0.47
        1.0 × 103 28.80 ± 0.10 0.34 28.56 ± 0.20 0.70

      Table 1.  Intra- and inter- assay reproducibility of SYBR Green I Real-time PCR.

      To assess the assay's performance in a clinical setting, 40 liver samples from sick ducklings suspected of duck hepatitis virus infection were analyzed using the developed RT-qPCR assay and a traditional single RT-PCR assay. All the samples were obtained from duck farms located in the Zhejiang and Anhui provinces. The samples were mixed and ground into homogenates. All the positive clinical samples that were detected RT- qPCR assay were confirmed by DNA sequencing of the single RT-PCR products using the same specific primer. The DHAV-infected liver samples were characterized by sequencing the VP1 gene. The results of the two assays were 100% consistent, and both methods showed that 16 of the examined samples contained DHAV-1, while 24 contained DHAV-3.

      Outbreaks with DHAV-1 and DHAV-3 at the same areas have recently been reported in China and South Korea (Gan et al. 2014; Soliman et al. 2015; Lin et al. 2016; Zhang et al. 2017; Wen et al. 2018). Since antibodies to DHAV-1 and DHAV-3 have almost no cross-neutralization ability, only ducklings injected with the genotype matched vaccine will receive protection. (Kim et al. 2007). Therefore, the new assay meets the clinical requirements for an accurate, rapid, sensitive and reliable method to detect and distinguish DHAV-1 and DHAV-3. Although, several diagnostic methods have been developed to detect and distinguish DHAV-1 and DHAV-3 based on RT-PCR and RT-qPCR (Yang et al. 2008; Huang et al. 2012; Chen et al. 2013; Hu et al. 2016; Chen et al. 2019).The previous differentiation methods required at two primer pairs, increasing the experiment's complexity. We have developed and validated a SYBR Green I real time RT-qPCR assay, which uses a single unlabeled primer pair to amplify a conserved region of 5' -UTR of DHAV.

      Our experiments demonstrated that, the primer pair in this assay was able to detect virus samples in a range of concentrations. Also, this assay has no cross-reactivity DAstV, AIV, DRV, DEV, or DPV. Importantly, this diagnostic method can also distinguish DHAV-1 and DHAV-3 using MCA, based on the different Tm values for each genotype's amplicons. The results obtained by this diagnostic method were reproducible and can be easily applied to the clinical settings.

      In conclusion, in this study, we developed an MCA- based SYBR Green I RT-qPCR assay to detect and differentiate DHAV-1 and DHAV-3. This new assay is rapid, sensitive, specific, cost-effective and easy to conduct. To our knowledge, this is the first description of a multiplex RT-qPCR that can detect and distinguish DHAV-1 and DHAV-3 in the same tube using one primer pair.

    • This work was supported by the National Natural Science Foundation of China Grants 31300141 (to CCM) and Shanghai Key Laboratory of Veterinary Biotechnology Grants klab201702 (to CCM).

    • The authors declare that they have no conflict of interest.

    • This research was approved by the Ethics Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The international guidelines for the care and use of animals have been followed.

    Figure (1)  Table (1) Reference (20) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return